I was reading about non-homologous end joining (NHEJ) in my molecular biology of the gene textbook but the explanation provided in the text was rather vague to me, and I was not able to understand it completely. I understood that NHEJ can be used as a repair mechanism when double-strand breaks in DNA occur. The two broken ends are then joined together. What I didn't understand is how this is done. The two broken ends won't always have complementary bases so, won't this lead to mismatched bases? How is this problem fixed? I really need a detailed explanation of how NHEJ occurs please.

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Non-homologous end joining indeed induces errors in the affected sequence. But you have to keep in mind, that NHEJ is an emergency repair mechanism which involves a "repair or die" chance. If the chromosomal break is not repaired it is not unlikely that the cell will get into apoptosis, or, even worse, develops into a cancer cell. Introducing small errors is a relatively small price compared to this, although this can still disrupt genes or regulatory sequences. For the mechanism have a look onto the figure (from the first reference):

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For further reading have a look onto the Wikipedia article on NHEJ and/or the following references:

  1. The mechanism of human nonhomologous DNA end joining.
  2. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway.

NHEJ is indeed error prone. It is called "non-homologous" because it does not use a "homologous" template from another sequence-matching piece of DNA to guide the repair. Homologous repairs avoid causing mutations because the similar string of DNA acts as a template so that the cell knows what letters to put into the gap. When there's no template, there's no way for the repair machinery to know what specific letters to use...hence the mutations in NHEJ.

Interestingly, this NHEJ phenomenon is used to drive the current state-of-the-art in genetic engineering: CRISPR/Cas9 RNA-guided endonucleases. If you cause a double-strand DNA break near the beginning of the protein-coding sequence of a gene, this break will be repaired with NHEJ, causing an "indel" (insertion or deletion) mutation to occur. Inserting or deleting letters into the coding sequence of a gene will cause a "frame shift" where the 3-letter codons which tell the cell which amino acids to use get all jumbled up because the normal 3-3-3-3-3-3-3 pattern is disturbed. In this way, we can turn off genes that we want to target: just cause a frameshift in that gene with CRISPR.

If we want to INSERT something using genome editing, we need "homology directed repair" -- we'll give the CRISPR alongside another string of nucleotides which matches the strings that flank the place we are cutting, with a new sequence in the middle. So when CRISPR makes the cut, our homologous template will stick to the sides of the cut and the center piece will provide the cell with a blueprint for the insertion or edit we want to make.

So, you'll see as you think about it that the "homology" which is missing from NHEJ is the strand used as a repair template, and by manipulating these natural DNA repair processes which may or may not use homologous templates, we have a possible method to turn on, turn off, edit or insert genes. This means NHEJ isn't just a biological process anymore: it is an engineering technique.

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